Project Summary/Abstract In the United States, someone experiences a stroke every 40 seconds. Axonal injury and dysfunction are responsible for much of the disability observed after stroke. The human brain comprises equal proportions of gray matter and white matter, and white matter is injured in most strokes. Casein kinase 2 (CK2) is a protein kinase expressed in brain, including in white matter, and is regulated by ischemia. Our preliminary results show that CK2 inhibition attenuates WM ischemic injury and prevents oxygen glucose-deprivation (OGD)-induced axonal injury and oligodendrocyte death. In addition, CK2 inhibition attenuates loss of axonal mitochondria, providing clear evidence that CK2 signaling inhibition protects WM from ischemia by preserving oligodendrocytes and mitochondrial integrity and function. CK2 expression is increased in many cancers and CK2 inhibitors are currently undergoing clinical trials in that field. As CK2 activity modulates the impact of ischemic injury on axonal recovery, we will use OGD as a model of stroke to investigate the mechanism(s) by which CK2 regulates axon function in a pure WM tract, the mouse optic nerve. The long-term goal of this application is to determine how CK2 signaling leads to WM ischemic injury. Our approach combines electrophysiology, biochemistry, RNA interference in oligodendrocyte cell lines, confocal and 2-photon imaging, tests of mitochondrial function, and 3-dimentional electron microscopy to test the hypothesis that CK2 activation of cyclin-dependent kinase 5 (Cdk5) and phosphatase and tensin homolog (PTEN)/AKT signaling pathways mediates WM ischemic injury by contributing to oligodendrocyte death and impairing mitochondrial function. This proposal is aimed to define which glial cell type is injured by CK2 signaling and to establish that CK2 signaling impairs mitochondrial dynamics, function, and ultrastructure. We anticipate that these studies will provide fundamentally important new insights into the physiological and pathophysiological regulation of CK2 signaling in WM. Our translational studies will identify novel therapeutic target(s) to protect WM against ischemic injury, reducing mortality and morbidity and improving recovery following stroke.